Notwithstanding the mass of information accumulated about cytochrome c, our knowledge of the potein mechanisms by which the structural properties and the functional activities of the molecule are sustained is still minimal. It is intended to employ recombinant DNA techniques to obtain cytochromes c varying from each other by single residues in all desired positions, so as to test directly and unambiguously the contributions of each sequence segment. Employing the cloned gene for iso-1 cytochrome c of baker's yeast as a molecular hybridization probe it is expected to isolate and clone the structural genes for cytochrome c in a variety of suitable species, including several vertebrates. The nucleotide sequences of these genes will be determined and in vitro directed mutagenesis techniques will be used to change single nucleotide base pairs as required. An expression system will be developed whereby the mutant genes can be introduced into suitable recipient cells, such as yeast cells incapable of making their own cytochrome c, the mutant cytochromes c isolated and studied for their structural, physico-chemical and enzymic activity properties. When these procedures become practical, it is intended to mutate in repeated single steps a vertebrate cytochrome c gene, such as the mouse gene, to its form in a common ancestor to another group of vertebrates, such as the primates, and then mutate it down the phylogenetic line leading to that other taxonomic group. Such experiments should make it possible to discern the probable path of evolutionary descent, examine the structural and functional properties of each intermediate form and determine whether evolutionary transitions are or are not always accompanied by functional effects which may be related to selective advantage. The primate portion of the statistical phylogenetic tree is a particularly interesting area in this regard, since the primitive primate, New World monkey, Old World monkey, ape and human cytochromes c have shown a remarkable series of functional changes accompanied by corresponding changes in their cytochrome c oxidases.